Biology subjects

Abraham, W.

Publications and source records attributed to Abraham, W..

12 recordsLinked to original sources

bioRxiv2026

Chronic subthreshold intermittent theta burst stimulation promotes structural axon initial segment plasticity in cortical neurons

Repetitive transcranial magnetic stimulation (rTMS) is used widely in neuroscience to study and alter neural plasticity. The cellular mechanisms underlying the effect of rTMS on the brain remain unclear but is primarily thought to act via activity-dependent synaptic plasticity mechanisms. Here we investigated whether chronic repetitive magnetic stimulation in vitro and in vivo can induce another form of activity-dependent neural plasticity, axon initial segment (AIS) plasticity. Cortical neurons isolated from postnatal wild-type mice were stimulated with 6 hours of sham, repetitive magnetic stimulation in the form of intermittent theta-burst stimulation (iTBS), or 15 mM potassium chloride, with changes to AIS location and length measured +0 hours and +24 hours post-stimulation. In addition, adult transgenic mice expressing green fluorescent protein at the AIS received daily sham or iTBS over the primary motor cortices for 7 consecutive days and processed for microscopy 3 hours after the last stimulation. Analysis of neurons stimulated in vitro showed that chronic iTBS caused bidirectional and time-dependent shifts to the AIS position relative to the soma and a delayed shortening of the AIS length at +24 hours. In the adult mice, 7 consecutive days of daily iTBS decreased AIS lengths in layers 2/3 and 5 pyramidal neurons. Our findings provide in vitro and in vivo evidence that rTMS induces neuronal plasticity outside of the synapse, which may contribute to the long-lasting effect of rTMS on the brain with repeated stimulation protocols.

neuroscience↗

bioRxiv2026

Transregional astrocyte-dependent metaplasticity in the hippocampus

Learning-related synaptic plasticity is regulated by metaplasticity, which adjusts plasticity thresholds in an activity-dependent manner. We have previously described a heterodendritic form of metaplasticity whereby priming stimulation in stratum oriens (SO) inhibits subsequent long-term potentiation (LTP) in the neighboring stratum radiatum of the hippocampal CA1 region. Here, we report that this metaplasticity is, in fact, transregional, in that the SO priming stimulation also inhibits later LTP induction at dentate gyrus (DG) middle molecular layer (MML) synapses, both in vitro and in vivo. This effect operates across the hippocampal fissure since it occurs in the absence of CA3, thus highlighting a previously unappreciated reverse-direction and long-distance hippocampal crosstalk. Our findings demonstrate an essential role of astrocytes as SO priming elicited a sustained increase in the frequency of calcium (Ca2+) events in astrocytes in the DG MML, while the metaplasticity effect was blocked by calcium-buffering in MML astrocytes. It could be triggered by either activation of M1 muscarinic acetylcholine receptors or group II metabotropic glutamate receptors, and was critically dependent on inositol 1,4,5-trisphosphate receptor type 2 signaling. The transregional inhibition of LTP was mediated by astrocytic release of tumor necrosis factor (TNF), which likely acts in an autocrine fashion on astrocytic TNF type 1 receptors (TNFR1s). Downstream of TNF-TNFR1 signaling, the inhibition of MML LTP was mediated by the activation of GluN2B-containing N-methyl-D-aspartate receptors. Thus, a complex, bidirectional neuron-glia signaling cascade orchestrates long-distance metaplasticity across hippocampal subregions, providing a novel framework for understanding how hippocampal neuronal networks dynamically regulate plasticity thresholds across space and time. Significance StatementThis study documents the existence of a transregional metaplasticity that traverses from area CA1 to the dentate gyrus across the hippocampal fissure. This reveals a novel long-distance crosstalk within the hippocampus, in addition to the classical pathways of information transfer. This form of metaplasticity entails an essential contribution by astrocytes that integrate neuronal activity and use a complex Ca2+-dependent neuron-glia-neuron signaling cascade to dynamically regulate LTP thresholds in the hippocampal dentate gyrus.

neuroscience↗

bioRxiv2025

Targeted suppression of type 1 interferon signaling during RNA delivery enhances vaccine-elicited immunity

RNA vaccines have emerged as a breakthrough technology, and one promising modality employs alphavirus-derived self-replicating RNA (repRNA) to express vaccine antigens. However, both the lipid nanoparticles (LNP) commonly used to deliver RNA and virus-like amplification of repRNAs trigger innate immune recognition, especially via type I interferon (IFN) signaling. To modulate IFN responses during vaccination, we formulated LNPs co-delivering antigen-encoding RNA together with siRNA targeting the interferon-/{beta} receptor-1 (IFNAR1). siRNA-mediated repression of IFNAR1 increased antigen expression from repRNAs by >10-fold, increased immune cell infiltration, and increased antigen presenting cell activation in the injection site and draining lymph nodes. Compared to repRNA alone, siRNA/repRNA co-delivery increased serum antibody titers >10-fold, dramatically augmented antigen-specific germinal center (GC) B cell responses, and primed 4.4-fold more antigen-specific T cells. Ifnar1 silencing by siRNA co-delivery similarly enhanced mRNA vaccines. Thus, siRNA co-delivery is a readily translatable approach to substantially enhance the immunogenicity of RNA vaccines.

bioengineering↗

bioRxiv2025

The superior colliculus gates dopamine responses to conditioned stimuli in visual classical conditioning

In classical Pavlovian conditioning, it is well-established that midbrain dopamine neurons respond to conditioned stimuli (CS) that predict a reward. However, how the dopamine neurons associate a neutral CS to a reward remains unknown. Here, we show that the superior colliculus (SC) develops neuronal responses to a visual CS during conditioning, which in turn drive the responses of dopamine neurons. Visual responses in the SC were only potentiated when a behaviorally meaningful time interval separated the visual stimulus and reward. Potentiation also required the convergence of visual, dopamine and serotonin inputs to the SC. Importantly, blocking potentiation of the visual response was sufficient to suppress the dopamine responses following a CS. These results reveal a mechanism for how the brain forms associations between unconditioned stimuli and behaviorally meaningful visual information during classical conditioning.

neuroscience↗

bioRxiv2024

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR (FMC63) as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-ALL/Lymphoma, and enhanced control of disease progression. This approach thus enables vaccine boosting to be applied to any clinically relevant CAR-T cell product.

bioengineering↗

bioRxiv2024

Long-Term Potentiation Produces a Sustained Expansion of Synaptic Information Storage Capacity in Adult Rat Hippocampus

Long-term potentiation (LTP) has become a standard model for investigating synaptic mechanisms of learning and memory. Increasingly, it is of interest to understand how LTP affects the synaptic information storage capacity of the targeted population of synapses. Here, structural synaptic plasticity during LTP was explored using three-dimensional reconstruction from serial section electron microscopy. Storage capacity was assessed by applying a new analytical approach, Shannon information theory, to delineate the number of functionally distinguishable synaptic strengths. LTP was induced by delta-burst stimulation of perforant pathway inputs to the middle molecular layer of hippocampal dentate granule cells in adult rats. Spine head volumes were measured as predictors of synaptic strength and compared between LTP and control hemispheres at 30 min and 2 hr after the induction of LTP. Synapses from the same axon onto the same dendrite were used to determine the precision of synaptic plasticity based on the similarity of their physical dimensions. Shannon entropy was measured by exploiting the frequency of spine heads in functionally distinguishable sizes to assess the degree to which LTP altered the number of bits of information storage. Outcomes from these analyses reveal that LTP expanded storage capacity; the distribution of spine head volumes was increased from 2 bits in controls to 3 bits at 30 min and 2.7 bits at 2 hr after the induction of LTP. Furthermore, the distribution of spine head volumes was more uniform across the increased number of functionally distinguishable sizes following LTP, thus achieving more efficient use of coding space across the population of synapses. SignificanceEstablishing relationships between structure, function, and information storage capacity provides a new approach to assessing network strength from structural measurements. Long term potentiation (LTP) is a standard model for investigating synaptic mechanisms of learning and memory. Information is a retrievable quantity that is being stored in synapses as synaptic strength and is correlated with multiple structural components of synaptic strength. Structural synaptic plasticity was measured in 3D reconstructions from serial section electron microscopy of spine head volume, as a proxy for synapse strength, at 30 min and 2 hr after LTP induction. Outcomes indicate that LTP enhances information storage capacity for at least 2 hr by increasing the precision of the synaptic structure and expanding the range of synapse sizes.

neuroscience↗

bioRxiv2023

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons

Antigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LFN). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings.

immunology↗

bioRxiv2023

Astrocyte Ca2+ signalling mediates long-distance metaplasticity in the hippocampal CA1

Astrocytes play an increasingly recognised role in regulating synaptic plasticity, but their contribution to metaplasticity is poorly understood. We have previously described a long-distance form of metaplasticity whereby priming stimulation in stratum oriens inhibits subsequent LTP in the neighbouring stratum radiatum of the hippocampal CA1 region of both rats and mice. Using genetic and pharmacological strategies to manipulate astrocytic Ca2+ signalling, we now show this form of metaplasticity requires inositol triphosphate receptor-dependent Ca2+ release in these cells. Blocking Ca2+signalling or inositol triphosphate receptors in single radiatum astrocytes abolishes the metaplasticity at nearby synapses. We also show the relevant Ca2+release in astrocytes is driven by adenosine A2B receptors, and stimulation of these receptors elicits the metaplasticity effect both in vitro and in vivo. Further, the metaplasticity requires signalling via tumor necrosis factor, but this cytokine is required to act on astrocytes, not neurons. Instead, glutamate, acting on GluN2B-containing NMDA receptors, is the likely gliotransmitter that signals to neurons to inhibit LTP. Together these data reveal a novel role for astrocytes in hippocampal LTP regulation across broader spatiotemporal scales than previously recognised. Main pointsO_LIIn hippocampal CA1, "priming" activity inhibits subsequent LTP at synapses hundreds of microns away. C_LIO_LIThis effect requires astrocytic Ca2+signaling, and a molecular cascade involving adenosine A2B receptors, tumor necrosis factor and GluN2B-containing NMDA receptors. C_LIO_LIThe metaplasticity effect is evident in vitro and in vivo. C_LIO_LILong-distance astrocyte signaling is a mechanism for regulating neural activity over broad spatiotemporal scales. C_LI

neuroscience↗

bioRxiv2022

Eradication of tumors with pre-existing antigenic heterogeneity by vaccine-mediated co-engagement of CAR T and endogenous T-cells

Chimeric Antigen Receptor (CAR) T-cell therapy can be effective in treating human cancer but loss of the antigen recognized by the CAR poses a major obstacle. Here, we report an approach for vaccine boosting CAR T-cells, which triggers engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T-cells promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited priming of endogenous anti-tumor T-cells (antigen spreading). This process was accompanied by a shift in toward oxidative phosphorylation in CAR T-cells and was critically dependent on CAR-T-derived IFN-{gamma}. Antigen spreading induced by vaccine-boosted CAR-T enabled a proportion of complete responses even when the initial tumor was 50% CAR-antigen-negative, and heterogenous tumor control was further enhanced by genetically amplifying CAR-T IFN-{gamma} expression. Thus, CAR T-cell-derived IFN-{gamma} plays a critical role in promoting antigen spreading, and vaccine boosting provides a clinically-translatable strategy to drive such responses against solid tumors.

immunology↗

bioRxiv2021

Spatially regulated protease activity in lymph nodes renders B cell follicles a sanctuary for retention of intact antigens

The structural integrity of vaccine antigens is critical, as antigen degradation in vivo could eliminate neutralizing epitopes and create competing B cell responses against irrelevant breakdown products. Using FRET imaging and imaging zymography, we found that protease activity and antigen breakdown are spatially heterogeneous in lymph nodes. Following protein immunization, antigens are rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions of the tissue. By contrast, the follicles and follicular dendritic cell (FDC) networks exhibit low protease activity and antigen degradation rates. Immunization regimens targeting antigen rapidly to FDCs led to germinal centers (GCs) where responses to intact antigen were highly dominant, while traditional bolus immunizations led to weaker GC responses where more GC B cells bound to breakdown products than intact antigen. Thus, spatially-compartmentalized antigen proteolysis impacts humoral immunity and can be exploited to enhance vaccine-induced production of antibody responses against key pathogen structural epitopes.

immunology↗

bioRxiv2021

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2 and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non or weakly neutralizing ("off-target") antibodies. Using our recently developed electron microscopy epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

immunology↗

bioRxiv2020

The Injury Response to DNA Damage Promotes Anti-Tumor Immunity

Inhibition of immune checkpoints has shown promising results in the treatment of certain tumor types. However, the majority of cancers do not respond to immune checkpoint inhibition (ICI) treatment, indicating the need to identify additional modalities that enhance the response to immune checkpoint blockade. In this study, we identified a tumor-tailored approach using ex-vivo DNA damaging chemotherapy-treated tumor cells as a live injured cell adjuvant. Using an optimized ex vivo system for dendritic cell-mediated T-cell IFN-{gamma} induction in response to DNA-damaged tumor cells, we identified specific dose-dependent treatments with etoposide and mitoxantrone that markedly enhance IFN-{gamma} production by T-cells. Unexpectedly, the immune-enhancing effects of DNA damage failed to correlate with known markers of immunogenic cell death or with the extent of apoptosis or necroptosis. Furthermore, dead tumor cells alone were not sufficient to promote DC cross-presentation and induce IFN-{gamma} in T-cells. Instead, the enhanced immunogenicity resided in the fraction of injured cells that remained alive, and required signaling through the RIPK1, NF-kB and p38MAPK pathways. Direct in vivo translation of these findings was accomplished by intra-tumoral injection of ex vivo etoposide-treated tumor cells as an injured cell adjuvant, in combination with systemic anti-PD1/CTLA4 antibodies. This resulted in increased intra-tumoral CD103+ dendritic cells and circulating tumor antigen-specific CD8+ T-cells, leading to enhanced anti-tumor immune responses and improved survival. The effect was abrogated in BATF3-deficient mice indicating that BATF3+ DCs are required for appropriate T-cell stimulation by live but injured DNA-damaged tumor cells. Notably, injection of the free DNA-damaging drug directly into the tumor failed to elicit such an enhanced anti-tumor response as a consequence of simultaneous damage to dendritic cells and T-cells. Finally, the DNA damage induced injured cell adjuvant and systemic ICI combination, but not ICI alone, induced complete tumor regression in a subset of mice who were then able to reject tumor re-challenge, indicating induction of a long-lasting anti-tumor immunological memory by the injured cell adjuvant treatment in vivo.

cancer biology↗